Progesterone has two opposing biological actions during the structural and functional development of the pregnant mammary gland. A proliferative action stimulates ductal side branching and formation of lobuloalveoli. At the same time progesterone has the anti-differentiative effect of repressing milk protein gene expression until parturition. The primary hormone responsible for regulation of milk protein expression is prolactin whose effect is mediated through activating interaction of the Stat5 transcription factor with the promoters of milk protein genes. Glucocorticoids potentiate the effect of prolactin through a positive cooperative interaction of the glucocorticoid receptor (GR) with Stat5. Our preliminary in vitro positive cooperative interaction of the glucocorticoid receptor (GR) with Stat5. Our preliminary in vitro results indicate that progesterone inhibits beta-casein expression at the level of gene transcription through a direct interaction of the glucocorticoid receptor (GR) with Stat5. Our preliminary in vitro results indicate that progesterone inhibits beta-casein expression at the level of gene transcription through a direct interaction of the progesterone receptor (PR) at the beta-casein promoter that interferes with prolactin/Stat5 signaling. The goal of this proposal is to define the mechanism of this direct PR-dependent inhibition of beta-casein transcription in vitro and in vivo. In AIM #1, biochemical approaches will be used to define cooperative protein-protein and protein-DNA interactions between PR and Stat5 that contribute to PR-mediated inhibition of Stat5 activity, or to the potentiating effect of GR. In AIM #2, cell-based transcription assays in mammary epithelial cell cultures will be used to define the mechanism by which PR inhibits Stat5 and GR-dependent transcription of beta-casein reporter genes. Aim #3, we will determine whether mechanisms of PR- dependent inhibition of beta-casein gene transcription defined in vitro also occur in the mammary gland in vivo by analysis of regulatory elements of beta-casein reporter genes expressed by recombinant adenovirus. In AIM #4, we will determine the relative contribution of direct and indirect recombinant adenovirus. In AIM #4, we will determine the relative contribution of direct and indirect (paracrine) mechanisms of repression of beta-casein transcription by PR in vitro and in vivo. The expectation of this research is to define a mechanism of negative gene regulation by PR that is unique to milk protein genes and thus fulfills a specific biological role of progesterone in the mammary gland during pregnancy.